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Nuclear Extracts-Neonatal Cardiac

Michael S. Parmacek
Ref: adopted from K. Chien (personal communication)

Reagents:

1. PBS-ice cold

2. Buffer A (10 mM Hepes pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM PMSF):

500 ul 1M Hepes pH 7.9
75 ul 1M MgCl₂
500 ul 1M KCl
25 ul 1M DTT (add just prior to use)
250 ul 100 mM PMSF (add just prior to use)
48.5 ml ddH₂0

3. Extraction Buffer (20 mM Hepes pH 7.9, 25% glycerol, 0.55M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT,1 ug/ml antipain, 1 ug/ml chymostatin, 1 ug/ml leupeptin, 1 ug/ml pepstatin):

1. 1 ml 1M Hepes pH 7.9
2. 12.5 ml glycerol
3. 27.5 ml 1M NaCl
4. 75 ul 1M MgCl₂
5. 20 ul 500 mM EDTA
6. 25 ul 1M DTT (add just prior to use)
7. 250 ul 100 mM PMSF (add just prior to use)
8. 50 ul 1 mg/ml antipain (Sigma A6271 1mg $10.10. Make up day of extraction)
9. 50 ul 1 mg/ml chymostatin (Sigma C7268 1mg $12.70. Make up day of extraction)
10. 50 ul 1 mg/ml pepstatin (Sigma P4265 1mg $10.45. Make up day of extraction)
11. 50 ul 1 mg/ml leupeptin (Sigma L2884 1mg $10.10. Make up day of extraction)
12. 8 ml ddH₂0

4. Dialysis Buffer (40 mM KCL, 15 mM Hepes pH 7.9, 1 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, 20% glycerol):

1. 40 ml 1M KCl
2. 15 ml 1M Hepes pH 7.9
3. 2 ml 500 mM EDTA
4. 500 ul 1M DTT (add just prior to use)
5. 200 ml glycerol
6. 2.5 ml 100 mM PMSF (add just prior to use)
7. 740 ml ddH₂0

Methods:

1. 75-100 hearts of 1-4 d neonatal rats are divided into 3-4 pools of 25 hearts. Each pool is rinsed 3-4 times in ice cold PBS to get of blood cells.

2. 5 ml of Buffer A is added to each pool, and it is homogenized in a Dounce homogenizer using piston B until tissues are homogeneous. All homogenized tissues are then combined into a 50 ml Oak Ridge tube.

3. The homogenized tissues are centrifuged at 6000 RPM in a (pre-cooled) JA 13.1 rotor at 4°C for 15 min, and the supernatant is discarded.

4. The pellet is resuspended in 1 ml/10 hearts (7.5 mls for 75 hearts) and re-homogenized with piston B for 15-20 strokes.

5. The homogenized pellet is centrifuged at 6000 RPM, 4°C, 15 min, and the supernatant is discarded.

6. The nuclear pellet is resuspended in 1 ml/20 hearts (3.5 ml/75 hearts) of Extraction Buffer and homogenized with piston A for 20-30 strokes.

7. The homogenized nuclear pellet is transferred to several pre-cooled microfuge tubes and centrifuged in the microcentrifuge at 4°C at the highest speed for 30 minutes.

8. The supernatant is dialyzed in 1L dialysis buffer for 4 hours at 4°C.

9. The dialyzed nuclear extract is quick frozen in dry ice ethanol and stored at -70°C.

 

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